One of the most interesting phenomena associated with gene rearrangement in the immune system is allelic exclusion, in which individual B- and T-cells express one specific antigen-receptor molecule. In order to guarantee the production of a unique receptor-type per cell, the immune system includes mechanisms for restricting expression to only one out of the two possible alleles. The investigator's recent experiments indicate that in B-cells that contain one unmethylated rearranged gene, the germline allele is methylated, proving that the initial demethylation is monoallelic, and strongly suggesting that it is this modification that marks the allele for rearrangement and expression. However, it is not yet clear how one allele is marked for demethylation. In order to understand this process the investigator will identify the cis-acting elements and the trans-acting factors that are involved in demethylation of a single allele. The possibility that one allele is more accessible than the other will be investigated, and the role of chromatin structure and germline transcription in this process will be studied. A likely possibility is that the two alleles are actually marked during the initial stages of B-cell development or even earlier. This results in both chromosomes having a different local structure. In keeping with this idea, the kappa genes have been found to be located within an asynchronously replicating chromosomal domain. Thus, it is proposed to decipher the molecular mechanisms that underlie the process of asynchronous replication. With this information in hand, the investigator will study the possible link between replication, chromatin accessibility and methylation, and their role in kappa chain rearrangement during normal B-cell development and in allelic exclusion. This may be very similar to what happens in the process of X-chromosome inactivation, where asynchronous replication and methylation differentially mark the two chromosomes.